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forensicbiochem > EXAM3 : data interpretation > Flashcards

EXAM3 : data interpretation Flashcards

1
Q

what are the steps in STR genotyping / interpretation

A
  • peak detection/interpretation thresholds
  • peak sizing
  • DNA size (allele call)
  • DNA fragment analysis
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2
Q

what does internal size standard do?

A

it runs along with the sample and it shows the size vs time of DNA fragments

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3
Q

___ is the bin defined around each allele

A

+/- 0.5

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4
Q

what is allele call?

A

determined by comparison of
peak size (bp) to allelic ladder
allele peak sizes run under the
same electrophoretic conditions

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5
Q

what is the detection threshold ?

A

50-200 RFU

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6
Q

why do we use thresholds?

A

to separate signal from noise (is the peak real or not)

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7
Q

analytical RFU ____

A

50

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8
Q

Interpretation RFU

A

150 RFU

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9
Q

what is signal peak height measured in ? what is it related to?

A

Relative Fluorescence Units (RFU)
- related to the amount of DNA present in the sample loaded onto the analysis instrument

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10
Q

what 3 issues impact mixture interpretation?

A

1.) peak detection threshold
2.) peak height ratio
3.) stutter percentage

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11
Q

biological artifact vs technology artifact issues

A

B: stutter, “N” peak, Off ladder, Tri-Alleles
T: Pull up, Dye blobs, air bubbles, urea crystals/particles, and voltage peaks

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12
Q

what happens during allelic drop-outs?

A

degradation and inhibition of the primer binding site mutation

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13
Q

what are the data interpretation issues

A
  1. artifact peaks vs allele peaks
  2. allelic drop outs
  3. mixtures
  4. mutations
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14
Q

what are stutter products ?

A

peaks that show up primarily one repeat less than the true allele

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15
Q

how does stutter happen?

A

a result of strand slippage during DNA synthesis

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16
Q

which repeat regions generate more stutter?

A

longer

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17
Q

where is stutter less pronounced ?

A

larger repeat unit sizes

18
Q

true or false: each successive stutter product is less intense

A

true

19
Q

how much of the true allele in tetranucleotides repeats STR loci

A

5-15%

20
Q

(N-4) stutter product is …

A

Deletion - caused by slippage on the copied BOTTOM strand

20
Q

(N+4) stutter product is …

A

Insertion - caused by slippage of the copying TOP strand

21
Q

In strand slippage, the lower incorporation rate allows what?

A

more opportunity for the DNA strands to breathe apart during PCR (same mechanism as mutation)

22
Q

Taq Polymerase - stutter peaks

A

50-60 nt/second

23
Q

Stoffel fragment - stutter peaks

A

5-10 nt/second

24
Q

Is a “stutter” the same size as an allele ?

A

YES !

25
Q

what is the average stutter in D3S1358/ vWA?

A

7%

26
Q

what is the average stutter for TPOX?

A

3%

27
Q

percent stutter tends to increase with __

A

allele length

28
Q

the proportion of stutter to main allele is …

A

generall reproducible within a locus and even for a given allele length

29
Q

alleles with longer core repeat unit regions generally have _____ proportion of stutter

A

increase

30
Q

alleles with interrupted core repeat regions tend to have ___ proportion of stutter

A

less

31
Q

what are the strategies for stutter reduction?

A
  • cosolvents: osmolytes
  • cycling parameters
  • mutants of taq polymerase
32
Q

In non-template addition what does taq polymerase do?

A

it will often add an extra nucleotide to the end of a PCR product , often “A” (adenlyation)

33
Q

why is non-template addition dependent on the 5’-end of the reverse primer?

A

it is dependent on this end so that a “G” can be put at the end of a primer to promote the non-template addition

34
Q

how can the non-template addition be enhanced?

A

with extension soak at the end of the PCR cycle ( 15-45 min @60 or 72 degrees to give polymerase more time)

35
Q

excess amounts of DNA template in the PCR reaction can result in…

A

incomplete adenylation (not enough polymerase to go around)

36
Q

why do we NOT want a mixture of (+/- A peaks)

A

to avoid split peaks

37
Q

What observations indicate that a peak in an electropherogram is a
spike as opposed to DNA?

A

sharp peak, that shows up in all of the panels (spike), where the allele is more curved

38
Q

Is it better to prevent or promote non-template addition? Explain your
reasons?

A

promote, to get it to A+ , we want it to be A+!!

39
Q

What steps are typically taken to confirm the presence of an off-
ladder allele?

A

does not match up with allelic ladder, meaning it doesn’t show up in the ladder
** if it is off then you would run it again, and again
- sometimes there is a temperature difference in your sample vs ladder which would mess up with alignment
- if you keep seeing it then it is off ladder

40
Q
A

forensicbiochem (11 decks)

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